Protein biosynthesis by the ribosome proceeds in defined phases of initiation, protein elongation, termination, and ribosome recycling (Schmeing 2009a). Understanding the molecular mechanism of translation requires high-resolution descriptions of the motions in the ribosome that enable key translational events (Munro 2009; Schmeing 2009a; Dunkle 2010). A ratchet-like rotation of the small ribosomal subunit relative to the large ribosomal subunit (Frank 2000) is crucial to the positioning of tRNAs in intermediate—or hybrid—binding sites, in which the 3′-CCA termini and acceptor stems of tRNA advance by one site on the large subunit while the anticodon elements of tRNA remain fixed on the small subunit (Moazed 1989). Binding of tRNAs in hybrid sites is central to mRNA and tRNA movements on the ribosome when they are translocated after each peptide bond is formed, during termination, and during ribosome recycling (Semenkov 2000; Zavialov 2003). However, the molecular basis for ribosome positioning of tRNAs in hybrid sites has been unclear.
Atomic resolution x-ray crystal structures of the bacterial ribosome with ligands bound have revealed molecular details of conformational rearrangements taking place in the unratcheted ribosome (Schmeing 2009a). The first molecular descriptions of intermediate states of ribosome ratchet-like rotation at atomic resolution were provided by x-ray crystal structures of the Escherichia coli 70S ribosome (Zhang 2009), with additional sub-steps proposed based on cryo-EM reconstructions (Fischer 2010). A post-translocation rotated state of the ribosome was recently identified by cryo-EM (Ratje 2010), in a conformation similar to that of the Saccharomyces cerevisiae 80S ribosome in the absence of bound substrates (Ben-Shem 2010).
After the termination of protein synthesis, ribosome recycling is required to free ribosomes from the mRNA transcript to enable further rounds of translation. In bacteria and organelles, ribosome recycling factor (RRF) binds in the tRNA binding cleft of the 70S ribosome at the interface of the large (50S) and small (30S) subunits and interacts with the 50S subunit peptidyl transferase center (PTC) (Lancaster 2002; Agrawal. 2004). In so doing, RRF sterically occludes deacylated tRNA binding in the peptidyl-tRNA site (P site, P/P configuration) to favor tRNA positioning in the hybrid peptidyl/exit tRNA binding site (P/E configuration) (FIG. 1A) (Gao 2005; Sternberg 2009). In the P/E configuration, tRNA is bound simultaneously to the P site of the small (30S) subunit and to the E site of the large (50S) subunit (Moazed 1989). Binding of the GTPase elongation factor-G (EF-G) to the RRF-ribosome complex and subsequent GTP hydrolysis lead to the dissociation of ribosomal subunits (Savelsbergh 2009).
Using single-molecule fluorescence resonance energy transfer (smFRET) techniques to interrogate the conformational states of the ribosome, conditions were found that favored positioning the tRNA in the hybrid P/E configuration for longer periods and allowed crystallization of the ribosome with tRNA bound in the P/E hybrid site. From these crystals, the structure of the intact E. coli 70S ribosome at a resolution of ˜3.2 Å was determined and showed that the crystals contain two independent copies of the ribosome per asymmetric unit in a “top-top” polysome configuration (Brandt 2009), with one ribosome in an unrotated state with the classic positioning of the tRNA at the P/P site and, as heretofore never crytallographically observed at this resolution, with the second ribosome in a fully-rotated state and having the tRNA bound in the hybrid P/E configuration, revealing new targets on the ribosome for drug discovery.
Many antibiotic agents in clinical use target bacterial protein synthesis. The majority of these chemically diverse compounds inhibit translation by targeting functional centers in the ribosome (Blanchard 2010). Despite the enormous size and complexity of the 2.5 Megadalton ribosome particle, only a few target sites have been identified: near the Peptidyl- (P) tRNA binding and messenger RNA (mRNA) decoding sites of the small ribosomal subunit, and near the peptidyl transferase and GTPase centers of the large ribosomal subunit (Poehlsgaard 2005; Tenson 2006). The ability to identify new target sites within this dynamic macromolecular machine depends on sensitive methods for their identification and validation (Llano-Sotelo 2009; David-Eden, 2010).
Aminoglycoside antibiotics in the 2-deoxystreptamine family are broad spectrum bacteriocidal agents used to treat gram-negative bacterial infections. In vivo, these compounds are thought to act by altering the mechanism of aminoacyl-tRNA (aa-tRNA) selection during mRNA decoding on the ribosome (Fourmy 1996; Rodnina 2000; Schmeing 2009a). Aminoglycosides do so by inducing local rearrangements in ribosomal RNA (rRNA) within the highly conserved helix 44 (h44) decoding site of the small (30S) subunit that allow near- and non-cognate tRNAs to be inappropriately recognized and incorporated into the Aminoacyl- (A) tRNA binding site. Increased levels of aa-tRNA mis-incorporation eventually exceed the cell's capacity to cope with the reductions in translational fidelity, ultimately leading to cell death (Zaher 2009). However, in vitro, these aminoglycosides inhibit a range of steps in the translation mechanism. This includes mRNA and tRNA translocation, the directional movement of substrates with respect to both subunits of the intact (70S) ribosome (Gale 1981; Feldman 2010), and ribosome recycling, the process of subunit separation following the termination phase of protein synthesis (Hirokawa 2002). The physical origins of these aminoglycoside-induced effects are not known.
Early biochemical studies demonstrated that aminoglycosides can bind to regions of the ribosome outside the canonical decoding region (Davies 1968; Dahlberg 1978). Recently, the aminoglycoside neomycin was shown crystallographically, on classic ribosomal structures, to bind to the bacterial ribosome within Helix 69 (H69) of 23S ribosomal RNA (rRNA) in the large (50S) subunit (Borovinskaya 2007). While binding at this site was proposed to be responsible for inhibition of ribosome recycling and possibly translocation, the structural refinement at the time was insufficient to identify the points of contact and interaction (Feldman 2010; Borovinskaya 2007). Notably, neomycin concentrations higher than 100 nM inhibit translocation as potently as the most effective translocation inhibitor known, viomycin (Feldman 2010). Ribosome complexes bearing the well-established A1408G neomycin-resistance mutation in the small subunit ribosomal RNA (rRNA) which disrupts neomycin binding to the h44 decoding site (Recht 1999) exhibit a similar translocation inhibition profile at the higher (micromolar) neomycin concentrations, suggesting that strong inhibitory effects arise from the binding of neomycin outside of the canonical h44 decoding region (Feldman 2010).
Using the crystals reported herein with a combination of smFRET and further detailed x-ray crystallographic methods, the molecular basis of neomycin-induced inhibition of translation processes specific to its interactions with H69 of the large subunit have been determined and reveal the complete structural extent of a neomycin-binding pocket on the ribosome when neomycin stabilizes an inactive configuration of the ribosome. This site is termed the H69 neomycin-binding site or pocket and provides a powerful starting point for rational drug design.